Separation Science—The State of the Art of Biopharmaceutical Analysis: A Virtual Symposium

Learn about the latest practical techniques in biopharmaceutical analysis using separation science.

Register Free: https://www.chromatographyonline.com/lcgc_w/separation

Event Overview:

LCGC International is proud to present another edition of its successful virtual symposium series: Separation Science—The State of the Art of Biopharmaceutical Analysis: A Virtual Symposium.

Biopharmaceuticals—from monoclonal antibodies and antibody–drug conjugates to therapeutic oligonucleotides and messenger RNA (mRNA)—are among the most structurally complex medicines ever developed, and characterizing them demands increasingly sophisticated analytical tools. This virtual symposium brings together leading scientists from academia and industry to share the latest advances in separation science for biopharmaceutical analysis.

Presentations will cover the fundamentals of ion-pair reversed-phase liquid chromatography (IP-RPLC) for therapeutic oligonucleotides, including the separation of closely related impurities and phosphorothioate diastereomers; a multi-criteria approach to optimizing liquid chromatography–mass spectrometry (LC–MS)-based mRNA sequence mapping; and denaturing ion-pair hydrophilic interaction liquid chromatography (HILIC) and reversed-phase approaches for impurity characterization of fatty acid–conjugated small interfering RNAs (siRNAs). Speakers will also review affinity chromatography for the selective analysis and isolation of biopharmaceuticals and show how capillary isoelectric focusing–mass spectrometry (cIEF–MS) and cIEF fraction collection reveal the charge heterogeneity of monoclonal antibodies, antibody–drug conjugates, and antibody fragments. Together, these talks offer a practical, forward-looking view of how analytical scientists are meeting the challenges of today’s most complex therapeutic modalities.

Attendance is free. Don’t miss this opportunity to learn from leading practitioners in separation science and push your skills forward.

Agenda:

Morning Session: 9:00 AM EST | 2:00 PM GMT | 3:00 PM CEST

9:00 AM EST Introduction
Alasdair Matheson, Executive Editor, LCGC International

9:15 AM EST Therapeutic Oligonucleotide Separations: From Fundamental Principles to Analytical and Preparative Applications
Torgny Fornstedt, Professor of Analytical Chemistry, Karlstad University

Therapeutic oligonucleotides are challenging to separate because of their high charge, structural complexity, chemical modifications, and numerous closely related impurities. This presentation provides an overview of the separation science underlying their analysis and purification, with an emphasis on ion-pair reversed-phase liquid chromatography (IP-RPLC). Recent studies will show how stationary-phase properties, mobile-phase composition, ion-pairing reagents, temperature, and gradient conditions influence retention, selectivity, and efficiency. The mechanisms governing selectivity between oligonucleotides and closely related impurities, including phosphorothioate diastereomers, will be examined. The presentation will also show how mechanistic understanding and chromatographic modeling support rational method development and preparative purification and will briefly address peak integration and impurity quantification.

9:35 AM EST A Multi-Criteria Approach to Optimizing LC–MS-Based mRNA Oligonucleotide Mapping
Athanasios Tsalmpouris, PhD Candidate in Pharmaceutical Analysis, Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva

Messenger RNA (mRNA) has emerged as a major therapeutic modality, but the size and structural complexity of mRNA transcripts present significant analytical challenges. Sequencing technologies verify the primary sequence but cannot directly detect chemical modifications, whereas bottom-up liquid chromatography–tandem mass spectrometry (LC–MS/MS) mapping measures nucleotide masses directly. We present a multi-criteria framework benchmarking ion-pair reversed-phase liquid chromatography (IP-RPLC) and hydrophilic interaction liquid chromatography (HILIC) conditions using chromatographic and mass spectrometric metrics. Applied to three mRNA transcripts with different nucleosides, IP-RPLC methods using fluoroalcohols remained the gold standard, achieving sequence coverage above 90% after stringent filtering. This approach supports the rational selection of optimal LC–MS conditions for mRNA sequence mapping.

9:55 AM EST Expanding the Analytical Toolbox: Denaturing Ion-Pair HILIC and RP Approaches for Impurity Characterization of Fatty Acid–Conjugated siRNAs
Martina Lioi, Senior Expert Analytical Science, xRNA Analytical Development and Operations, Technical Research and Development, Analytical Research and Development (ARD), Novartis AG

Chemically modified small interfering RNAs (siRNAs) offer enhanced stability, binding affinity, and pharmacokinetic performance, and fatty acid (FA) conjugation has emerged as a promising route to extrahepatic delivery. However, FA conjugation creates a polarity mismatch within the siRNA duplex, demanding highly selective, orthogonal methods for purity and impurity analysis under denaturing conditions. Conventional hydrophilic interaction liquid chromatography (HILIC) may not fully denature duplexes, while ion-pair reversed-phase (IP-RP) retention can be dominated by the FA moiety. Ion-pair HILIC (IP-HILIC) can increase selectivity and promote duplex denaturation. This presentation evaluates denaturing ion-pair separations in RP and HILIC modes for FA-conjugated siRNAs, confirming the complementary strengths of IP-HILIC and IP-RP.

10:15 AM EST Affinity Chromatography for Biopharmaceutical Analysis: An Overview
David S. Hage, Hewett University Professor of Chemistry, Department of Chemistry, University of Nebraska–Lincoln

Biopharmaceuticals represent a new set of challenges for bioanalytical chemistry and separations. Affinity chromatography offers many attractive features for the selective analysis or isolation of these agents based on their structures or biological activities. This approach uses an immobilized, biologically related binding agent as a stationary phase to capture or measure specific targets. This presentation will discuss the principles of affinity chromatography as a tool for bioanalysis, with an emphasis on its use with biopharmaceuticals and related compounds. Examples will include methods that use antibodies, antigens, and immunoglobulin-binding proteins as stationary phases, as well as other binding agents. Applications ranging from biopharmaceutical analysis to sample pretreatment and analyte characterization will also be discussed.

10:35 AM EST Characterizing Antibody Heterogeneity Through cIEF–MS and cIEF Fraction Collection
Jasmin Schairer, Associate Scientist, RIC Group

Capillary isoelectric focusing (cIEF) is widely used to evaluate protein and antibody charge heterogeneity, but peak identification has traditionally been labor-intensive. Recent advances in ampholytes, cartridges, and instrumentation now enable the direct coupling of cIEF with mass spectrometry (MS) and the collection of cIEF fractions. These innovations will be illustrated using monoclonal antibodies (mAbs), antibody–drug conjugates (ADCs), and antibody fragments. Direct cIEF–MS enables sensitive, rapid molecular weight determination of charge variants, while collected fractions are assessed by bottom-up liquid chromatography–mass spectrometry (LC–MS), enzyme-linked immunosorbent assay (ELISA), and nano differential scanning fluorimetry (nanoDSF). Operating cIEF in normal or reverse mode controls the mobilization order of variants, improving the characterization of low-abundance species.

10:55 AM EST Question and Answer Session
The panelists will answer your questions on biopharmaceutical analysis.

Afternoon Session: 12:00 PM EST | 5:00 PM GMT | 6:00 PM CEST

Experts from leading chromatography companies will describe how new chromatography technologies can improve your methods in practice.

Speakers:

Torgny Fornstedt
Professor of Analytical Chemistry
Karlstad University

Athanasios Tsalmpouris
PhD Candidate in Pharmaceutical Analysis
Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva

Martina Lioi
Senior Expert Analytical Science
xRNA Analytical Development and Operations, Technical Research and Development, Analytical Research and Development (ARD), Novartis AG

David S. Hage
Hewett University Professor of Chemistry
Department of Chemistry, University of Nebraska–Lincoln

Jasmin Schairer
Associate Scientist
RIC Group

Register Free: https://www.chromatographyonline.com/lcgc_w/separation